STUDIES ON THE PRESENCE OF CREATININE IN

HUMAN BLOOD
BY BENJAMIN F. MILLER AND RENE DUBOS

(From the Hospital of The Rockefeller fnstitute for Medical Research, New
York)

(Received for publication, July 23, 1937)

Although the presence of creatinine in urine had been recognized by Liebig

in 1847 it was not until 1910 that evidence was advanced for its existence in
blood. In that year Shaffer and Reinoso demonstrated that in protein-free
filtrates of blood it is possible to obtain a faintly positive Jaffe reaction similar
to that given by dilute creatinine solutions. This color reaction later became
the basis of the Folin (1914) method for the determination of
preformedcreatinine in blood. Later, Hunter and Campbell (1917) showed that
human erythrocytes contain a substance which is not creatinine but which
gives the typical Jade reaction. These authors did not, however, express any
doubt concerning the existence of creatinine in plasma.
In 1922 Behre and Benedict published an extensive series of observations on the
Jaffe-reactive material in blood. They compared the chromogenic’ substance in blood
filtrates with pure creatinine solutions or with filtrates containing added creatinine by (1)
treating with hot alkali, which destroys creatinine, (2) contrasting the colors produced
when sodium carbonate is substituted for sodium hydroxide in the Jade reaction, and (3)
adsorption studies with kaolin. These relatively non-specifi techniques elicited some
striking differences between pure creatinine and the chromogenic substance in blood
filtrates. Behre and Benedict concluded that “creatinine does not exist in blood in
detectable quantities.” This conclusion was supported by the similar experiments of
Gaebler (1930) and Bohn and Hahn (1933), but were not confirmed by those of Zacherl
and Lieb (1934),
Ferro-Luzzi (1935), Hayman, Johnston, and Bender (1935), and Danielson
(1936). The experiments by Danielson are of particular importance be-cause they
included a careful repetition of the several techniques employed by Behreand Benedict.
Danielson studiedboth ultrafiltratesand tungstic
The term chromogenic substance is used to indicate the material in blood
filtrates which yields the same reaction with alkaline picrate as given
by pure creatinine; it has the same connotation as “apparent creatinine” or
“Jaffe-reactive material.”
447

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 acid filtrates of plasma. His conclusion that “the greater part if not all
the chromogenic material in normal plasma is creatinine” is in sharp con-
rast to that of Behre and Benedict.
Benedict and Behre (1936) have not accepted Danielson’s conclusions,
and have presented additional evidence to support their original contention.
They demonstrated that a new reagent, dinitrobenxoic acid, is more specific
for creatinine than is alkaline picrate. They showed that although dinitro-
benzoic acid gives a typical “creatinine color” with urine, it yields an
entirely different shade in various types of blood and plasma filtrates.
Recently, however, Bolliger (1936) and Langley and Evans (1936) have sug-
gested methods with the dinitrobenzoic acid reagent for determination of
creatinine in blood filtrates. These investigators did not report any
unusual difficulties in obtaining a color match between blood filtrates and
pure creatinine standards.
In more recent experiments Behre and Benedict (1937) have used the
reaction between creatinine, rubidium chloride, and picric acid. This
reaction yields an insoluble creatinine rubidium picrate and may be em-
ployed to remove creatinine from pure solution. Applying this technique
to blood filtrates, Behre and Benedict were unable to obtain typical pre-
cipitates, although such precipitates occurred upon addition of pure cre-
atinine to the filtrates. These results seem of questionable importance for
the study of “creatinine” in blood in the light of experiments published
simultaneously by Gaebler (1937). This investigator found that a large
portion of the chromogenic material in plasma ultrafiltrates could be pre-
cipitated by the rubidium-picric acid reagent.
Evidence from physiological experiments has been sought by Goudsmit
(1936), who studied the extraction of the chromogenic compound of plasma
by the kidneys of the dog. He found an average value for the extraction
percentage which was almost identical with that obtained for exogenous
creatinine by Van Slyke, Hiller, and Miller (1935). However, Goudsmit
concluded that the “observations do not have a direct bearing on the
question of the nature of the chromogenic material in the blood.”

Our interest in the nature of the J&e-reactive material in human plasma

arose from the desire of one of us to investigate the renal excretion of
creatinine in man under normal physiological
conditions. In order to study the so called “endogenous creatinine clearance,”
it is essential to determine the nature of the chromo-genic material in plasma;
if this material proves to be creatinine,
a specific method must be available for its analysis. We felt that since similar,
and even identical, non-specific techniques had yielded entirely conflicting
results in different laboratories, they could not be relied upon to give a
definitive solution t.o this prob-lem. We therefore developed specific enzymes
for the decomposi-
 B. F. Miller and R. Dubos 449

tion of creatinine. By means of the two bacterial enzymes described

previously by Dubos and Miller (1937) it has been possible to study
the chromogenic material with a specific tech-
nique. In the present paper the action of the enzymes on the
chromogenic material in blood filtrates is compared with their action
on pure creatinine.
The study of the nature of the chromogenic compound by means of these
enzymes has been supplemented by the recently intro-
duced dinitrobenzoic acid reaction for creatinine. It has been found that
this reagent yields important evidence when it is used
with spectrophotometry instead of the usual calorimetric technique.

Effect of NC and HR Enzyme Preparations upon Jaffe-

Reactive Material of Blood and Spinal Fluid
In these experiments blood filtrates and spinal fluids were incu-
bated with the NC and HR enzymes. The Jaffe reaction was used to obtain the
total creatinine equivalent of these fluids before
and after incubation with the enzyme suspensions. In this way, it is
possible to estimate the amount of creatinine decomposed in the fluids.
The chromogenic material remaining after the en-zymatic destruction
of the creatinine is expressed in terms of its
creatinine color equivalent. This is necessary because the nature of
the residual Jaffe-reactive substances is unknown.

Methods
Blood and spinal fluid samples were obtained from normal, adult males. Most
of the observations were made upon tungstic acid filtrates prepared according to
the method of Wu (1922). Plasma samples were obtained
from oxalated blood. In the few instances in which spinal fluids were used
they were diluted with water or physiological saline.
An excess of the NC or HR enzyme was added to 10 cc. of the blood fil-trate or
spinal fluid containing 0.5 cc. of M phosphate buffer, pH 7.0 (made according to
Green (1933)). The requisite amount of cells was suspended
in 1 to 2 cc. of Hz0 or 0.9 per cent NaCl solution. The strength of the
enzyme preparation had been determined by the titration previously de-
cribed by Dubos and Miller (1937). The mixture was incubated in 125 cc.
nstoppered flasks at 37” for 30 to 90 minutes, the exact time depending
pon the strength of the enzyme preparation. The cells were removed by
centrifugation at high speed. To 8 cc. of the supernatant fluid were added 4 cc. of
alkaline picrate, and the resultant color was read after 10 minutes.
450 Creatinine in Blood
The analyses were all performed in a Zeiss Pulfrich stufenphotometer, with a
stratum thickness of 30 mm. and the 550 light filter. A control solution of water,
buffer, and enzyme was incubated, centrifuged, and treated with
the alkaline picrate. This served as the balancing solution in the stufen-photometer
to compensate for any turbidity and for the absorption of light
by the alkaline picrate reagent. The residual chromogenic substance was
determined as its creatinine equivalent from a calibration chart, and com-pared
with the value obtained for the total creatinine equivalent of the
original solution. (Further details regarding technique and accuracy of the
method are given in the subsequent paper.)

TABLE I
Amounts of Chromogenic Material Decomposed by Enzymes* in Serum,
Plasma, Erythrocytes, and Spinal Fluid from Normal Individuals
No. of
A-y
Material ,ietermi- chromo-
nations genie
material
--

?% 2
Serum or plasma. ................. 15 1.09
Erythrocytes ...................... 6 1.80
Spinal fluid. ...................... 2 1.11

* Most of the determinations were performed with the NC enzyme prep-

aration; but at least one analysis on each type of material was made with the
HR preparation.

Results
Filtrates of Serum and Plasma-Filtrates of normal human serum and
plasma were studied according to this technique. Most of the
determinations were made with NC enzyme prepa-rations, and a-smaller
number with HR cell suspensions. Table I indicates that an average of 91
per cent of the chromogenic material originally present was decomposed
by the enzymes. (The details of these analyses and those for the other
fluids are recorded in the following paper.)
Filtrates of Erythrocytes-In filtrates of erythrocytes there was an
average of 40 per cent decomposition of the original chromo-genic
material (Table I). When cells and plasma from the same blood were
analyzed, it was found that the amount of the cellular chromogenic
substance decomposed was equivalent to about three-fourths of that in
the plasma. This represents the same
 R, F. Miller and R. Dubos 451
ratio as that which would be calculated from the distribution of
water between cells and plasma (Van Slyke et al. (1925)).
Spinal Fluids-Since only small amounts of spinal fluids were
available, six to eight samples were pooled and analyzed. Table I shows
that the chromogenic material in these representative samples was as
readily decomposed by each of the enzyme prepa-rations (100 per cent!
in the two pooled samples available) as the material in plasma and
serum filtrates.

TABLE II
Concentrations Equivalent to CTeatinine Obtained by Piuric Aoid and
DinitTo-benzoic Acid Methods with Various Compounds Closely
Related to CTeatinine
Concentration e uivalent to
creatinine divide % by concon-
t&ion of compound
Compound -
I Xnitrobeneoio
acid method

Creatinine ..................................... 1.0 1.0

Glycocyamidine............................... 0.48 0.14
5-Methylglycocyamidine . ...................... 0.55 0.15
Methylcreatinine.............................. 0.48 0.12
Bensoylcreatinine.............................. 0.19 0.075
Acetylcreatinine............................... 0.16 0.18
Dimethylcreatinine ............................ 0.69 0.12
-
Solutions containing 0.01 mg. per cc. of each compound were compared by the two
methods; their colors were determined in a stufenphotometer.
The “creatinine equivalent” is the value for the light absorption of the various
solutions expressed in terms of creatinine.

Observations on Body Fluids by Means of Dinitrobenzoic Acid

Reaction
In these experiments the creatinine color equivalent of the chromogenic
material in blood filtrates was obtained by the dinitrobenzoic acid reagent
and compared with the concentration derived from the reaction with
alkaline picrate (Jaffe’s reaction).

Methods
The materials and fiNrates were obtained and prepared as described in the
previous section.
The method described by Langley and Evans was followed, including the
 Creatinine in Blood
use of the purified dinitrobenzoic acid reagent. However, a Zeiss Pu&-ich
stujenpholomeler WQSemployed in place of acolorimeler. The stufenphotom-etcr was
calibrated with pure creatinine solutions containing O.CK.lO25mg. to 0.005 mg. per cc.
When’the 30 mm. cups and the S50 light filter of the instrument were employed,
excellent checks for duplicate analyses could be
obtained in this range of creatinine concentration. The results conform
withthe Lambert-Beer law in this range, but not at higher concentrations. The
creatinine equivalents of glycocyamidine, 5-methylglycocyamidine,
and other related compounds were obtained by both the dinitrobenzoic acid

TABLE III
Valuesfor Crealinine Equivalent of Chomogenic Material inPlasma Pillrates
Obtained from Simultaneous Analyses by Alkaline Picrate and Dinitro-
benzoic Acid Melhods (Mg. per 100 Cc. of Original Fluid)
- -
ld5-tungBtat.~teS~anolvlcrlllnlpraated.
It Crentinine
concentmtion
Sample I(,ydinitroben-
zoate method
-- -I-

Plasma (nom& adult). . 108 1.10

“ ‘I ‘I ... . . . .. . 1.16 1.17
“ (pooled, normal adult). .. 1.03 1.02
“ ‘I “ I‘ .... .. . 0.91 0.85
‘I I‘ ‘I I, . 1.05 1.10
“ I( ‘I “ ................. 0.97 0.94
I‘ “ ‘I ‘I 0.94 0.91
” (adult with hypertension) 1 : : : : : : : : : 1: 1.29 1.23
‘I (nephritis). . . . 2.85 2.80
‘I I‘ ... . . . .... . . . 4.00 3.87
Plasma* (pooled, adult). . . .. 1.40 1.43

Spinal fluid.. 0.94 - 1.00

* Ultrafiltrate.

and picric acid reagents. The results are given in Table II. They
demonstrate
in almost every case that creatinine may be distinguished
from related chromogenic compounds more specifically by means of the dinitro-
benzoic acid method. Similar results with pure solutions were obtained
byBenedictandBehre.
Analysts by the two methods have been performed simultan-eously on the
chromogenic material of plasma and serum filkatcs and other fluids. The
analytical results are compared in Table
III. It is shown that in every sample the two values for the creatininc
equivalent of the chromogenic material agree very closely.
B. F. Miller and R. Dubos
DISCUSSION

These experiments indicate that creatinine exists in blood and

constitutes a large portion of the Jaffe-reactive material, espe-
cially in t.hc plasma. Each of the enzymes employed in this study posscsscs a
high degree of specificity toward creatininc, as con-
trasted with other Jaffe-reactive compounds. It seems likely, thcrcfore,
that that portion of the chromogenic material in blood
which is decomposed by the enzymes is also true crcatinine. The previous
study of the Jaffc-reactive compounds closely related to
crcatinine has shown that glycocyamidine is the only one of them
acted upon by both enzymes. However, the rate of decomposi-tion of
this substance is very much slower than that of creatinine.
That glycocyamidine is not the chromogenic compound in blood is
shown by the following evidence: (1) Experiments2 were per-formed
in which serum filtrates were incubated with the enzyme
and compared simultaneously with equivalent concentrations
(according to the Jaffe color) of pure glycocyamidinc. It was found
that decomposition of the chromogenic material in blood filtrat.es
proceeds much more rapidly than the decomposition of
glycocyamidine solution. (2) The results given for the com-
parison of the alkaline picrate and dinitrobenzoic acid reactions
in body fluids practically eliminate glycocyamidinc from serious
consideration. Even if low concentrations of glycocyamidine were
present they would seriously affect the creatinine equiva-
lents, especially those obtained by the dinitrobenzoic acid
method. Since in cvcry case the two methods gave the same
conccntrntion of creatinine, it is apparent that the chromogenic
material is not glycocyamidine or a mixture of glycocyamidine and
creatininc.
Since ccrt,ain non-chromogenic compounds such as creature, arginine, and
the guanidincs yield urea in the presence of the enzymes, it has not been
possibltt to compare the urea equivalent of the chromogenic subst ante in the
filt.rates with pure creatinine
solutions. The presence of unknown quantities of these other substances
which combine with the enzyme also makes it impos-siblc to compare the rate
of decomposit.ion of the chromogenic

2 These experiments were performed by the technique outlined in the first

section of “Methods.”
454 Creatinine in Blood
substance with equivalent amounts of pure creatinine. Such evidence
might be desirable but, in view of the extreme specificity represented by
the two distinct enzyme systems, it does not seem essential. Even if one
should grant the assumption that the NC
enzyme decomposes a chromogenic substance in blood in addition
to creatinine, it would appear extremely unlikely that the entirely
different HR enzyme would decompose creatinine and also the very
same chromogenic compound.
The analysis of the chromogenic substance of plasma by the
dinitrobenzoic acid reagent corroborates the results obtained by the
specific enzymes. This reagent gives a fairly specific color
test for creatinine. It is important that it yields approximately the
same values for the chromogenic substance as do the specific
enzyme methods with entirely different reagents.
Our results with the dinitrobenzoic acid reagent are not in agreement
with those obtained by Benedict and Behre. This diver-gence is probably
explained by Bolliger’s statement (1936) that “the inability of Benedict
and Behre to obtain in blood filtrates colours comparable with a suitable
standard lies in the fact that their dinitrobenzoic acid was not sufficiently
pure . . . impurities may give in the blank only a slight yellowish-brown
color reaction,
but they interfere most seriously with the determination of
creatinine in blood filtrates.” Langley and Evans in addition to Bolliger
obtained the dinitrobenzoic acid reaction in blood filtrates
and employed it to determine “creatinine.” Their results were very far
below those obtained by the alkaline picrate method, even when one
subtracts the non-creatinine chromogenic material. Our results show that
the dinitrobenzoic acid and the picric acid methods give close agreement
when used with the more precise
spectrophotometric technique. It seems reasonable that the low
values obtained by these workers were due to difficulties in their
calorimetric techniques.
That two color reactions, each with highly different sensitivities for
compounds closely related to creatinine, give the same cre-atinine values
in blood fihrates presents significant evidence for
the identity of the chromogenic material and creatinine. The more
specific results obtained with the two independent enzyme
preparations also lead to the same conclusion. That the exact
agreement obtained by the three procedures may be fortuitous seems
to us extremely unlikely.
It is assumed that the presence of creatinine in tungstic acid filtrates
of plasma or blood indicates the presence of the same compound in the
plasma or blood itself. It seems extremely
doubtful that the mild chemical manipulations involved in the
preparations of such filtrates would change a precursor into
creatinine. Furthermore, we have also shown that the same results may
be obtained with the chromogenic material in spinal fluid, which
represents a perfectly natural type of ultrafiltrate.
SUMMARY AND CONCLUSIONS

Experiments have been performed to determine the nature of the

chromogenic material (“apparent creatinine”) in human blood.
Two enzymes, each possessing a high degree of specificity for
creatinine as compared with other Jaffe-reactive compounds,
have been employed in this study. Each enzyme decomposes almost.
all the chromogenic material in normal plasma and about half of the
quantity found in erythrocytes.
A spectrophotometric method is described for use with dinitro-benzoate, a
reagent which reacts more specifically with creatinine
than does alkaline picrate (Jaffe’s reagent). This method indi-cates the
presence of creatinine in plasma, and also gives cre-atinine values
which are equivalent to those obtained by the alkaline picrate method.
Because of the specificity of the above methods, and the fact that
they all indicate identity of the chromogenic material and creatinine, it
is concluded that true creatinine exists in human blood. It is concluded
also that creatinine constitutes almost all of the chromogenic material
in normal plasma.
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